) The relationship between the chemistry of ATP hydrolysis and the mechanical events that result in rotation of the gamma subunit of the F1-ATPase will be examined. Catalytic function of F1 derives from the asymmetry of the catalytic sites that, in turn, depends on the gamma subunit and the Mg2+ cofactor. The driving force for gamma rotation is believed to result from the initial binding energy of the Mg2+-ATP complex and from the release of phosphate which is a Mg2+ ligand. The ability of the gamma subunit of F1 to rotate will be measured using a fluorescent microsphere attached to the gamma subunit as recorded using a CCD camera. The torque generated during gamma rotation as well as the dwell time between rotations will be as assessed with F1 that contains site-directed mutants or other treatments at locations that may affect the coupling between hydrolysis and gamma rotation. Three loci are targeted for investigation that include: (a) Switch 3, the gamma subunit C-terminus and the beta subunit greasy bearing which is close to the site of Mg2+ binding and phosphate release; (b) Switch 2, the interface between the gamma subunit and the beta subunit DELSEED sequence; and (c) Switch 1, where hydrogen bonds form between the gamma subunit and the betaE subunit catch loop. Experiments will examine the possibility that Switch 1 is part of an escapement mechanism that only allows gamma rotation when the catalytic sites are filled with metal-nucleotide complex. Experiments are also designed to identify changes in metal ligands at the catalytic sites that are specifically associated with conformational changes linked to gamma rotation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM050202-07
Application #
6636093
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Preusch, Peter C
Project Start
1996-08-01
Project End
2005-03-31
Budget Start
2003-04-01
Budget End
2004-03-31
Support Year
7
Fiscal Year
2003
Total Cost
$257,916
Indirect Cost
Name
Arizona State University-Tempe Campus
Department
Other Basic Sciences
Type
Schools of Arts and Sciences
DUNS #
943360412
City
Tempe
State
AZ
Country
United States
Zip Code
85287
Xiong, Fusheng; Frasch, Wayne D (2011) Padlock probe-mediated qRT-PCR for DNA computing answer determination. Nat Comput 10:947-959
Ishmukhametov, Robert; Hornung, Tassilo; Spetzler, David et al. (2010) Direct observation of stepped proteolipid ring rotation in E. coli F?F?-ATP synthase. EMBO J 29:3911-23
Spetzler, David; Ishmukhametov, Robert; Hornung, Tassilo et al. (2009) Single molecule measurements of F1-ATPase reveal an interdependence between the power stroke and the dwell duration. Biochemistry 48:7979-85
Hornung, Tassilo; Ishmukhametov, Robert; Spetzler, David et al. (2008) Determination of torque generation from the power stroke of Escherichia coli F1-ATPase. Biochim Biophys Acta 1777:579-82
York, Justin; Spetzler, David; Hornung, Tassilo et al. (2007) Abundance of Escherichia coli F1-ATPase molecules observed to rotate via single-molecule microscopy with gold nanorod probes. J Bioenerg Biomembr 39:435-9
Spetzler, D; York, J; Dobbin, C et al. (2007) Recent developments of bio-molecular motors as on-chip devices using single molecule techniques. Lab Chip 7:1633-43
Boltz, Kathryn W; Frasch, Wayne D (2006) Hydrogen bonds between the alpha and beta subunits of the F1-ATPase allow communication between the catalytic site and the interface of the beta catch loop and the gamma subunit. Biochemistry 45:11190-9
Spetzler, David; York, Justin; Daniel, Douglas et al. (2006) Microsecond time scale rotation measurements of single F1-ATPase molecules. Biochemistry 45:3117-24
Lowry, David S; Frasch, Wayne D (2005) Interactions between beta D372 and gamma subunit N-terminus residues gamma K9 and gamma S12 are important to catalytic activity catalyzed by Escherichia coli F1F0-ATP synthase. Biochemistry 44:7275-81
Boltz, Kathryn W; Frasch, Wayne D (2005) Interactions of gamma T273 and gamma E275 with the beta subunit PSAV segment that links the gamma subunit to the catalytic site Walker homology B aspartate are important to the function of Escherichia coli F1F0 ATP synthase. Biochemistry 44:9497-506

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